Rational Tuning of CAR Tonic Signaling Yields Superior T-Cell Therapy for Cancer

Abstract

SUMMARYChimeric antigen receptors (CARs) are modular proteins capable of redirecting immune cells toward a wide variety of disease-associated antigens. Here, we explore the effects of CAR protein sequence and structure on CAR-T cell function. Based on the empirical observation that CD20 CARs with similar sequences exhibit divergent tonic-signaling and anti-tumor activities, we devised engineering strategies that aimed to improve CAR-T cell function by tuning the intensity of tonic signaling. We found that CARs designed to exhibit low but non-zero levels of tonic signaling show robust effector function upon antigen stimulation while avoiding premature functional exhaustion by CAR-T cells. Through alterations of the CAR’s ligand-binding domain and overall protein conformation, we generated CD20 CAR variants that outperform the CD19 CAR in mouse models of human lymphoma. We further demonstrate that rational modification of protein confirmation can be generalized to improve GD2 CAR-T cell efficacy against neuroblastoma. These findings point to tonic signaling and basal T-cell activation as informative parameters to guide the rational design of next-generation CARs for cancer therapy.

SynNotch CAR circuits enhance solid tumor recognition and promote persistent antitumor activity in mouse models

“…Here, we focus on the development of more specific and potent next-generation engineered T cell therapies for mesothelioma and ovarian cancer. Mesothelioma is a highly aggressive chronic inflammationdriven cancer with an exceedingly poor prognosis that has three histological subtypes: epithelioid (69% of cases, median overall survival ~14 months), biphasic (12%, ~10 months), and sarcomatoid (19%, ~4 months) (7,8). Mesothelioma is inherently hard to treat with traditional modes of cancer therapy, and recent trials exploring mono-or combination immune checkpoint inhibitors (ICIs) have also had limited impact (10,11).…”

“…Tonic signaling is a function of the specific CAR architecture and high receptor cell surface densities that can facilitate receptor aggregation before antigen binding (5). Temporary suspension of CAR signaling or receptor redesigns have been proposed means to enhance antitumor activity by reduction of tonic signaling (6)(7)(8). Alternatively, tonic signaling can be reduced through targeted genomic integration into the TRAC locus, which allows for more controlled and dynamically regulated expression of CARs similar to the natural T cell receptor (9).…”

Charging CAR by electrostatic power

“…When the recyclable CAR design was applied to CD19‐CAR that is known to have weak tonic signaling, the in vivo persistence of CAR‐T was significantly increased, which was consistent with the Peak Theory. Interestingly, a research team from UCLA also published a preprint article on bioRxiv, 55 suggesting that the basal signal of CARs should be weak but not zero, which independently validates the “Peak Theory” from different perspectives.…”

“…Interestingly, a research team from UCLA also published a preprint article on bioRxiv, 55 suggesting that the basal signal of CARs should be weak but not zero, which independently validates the "Peak Theory" from different perspectives.…”

Engineering Next-Generation CAR-T Cells: Overcoming Tumor Hypoxia and Metabolism

“…Interestingly, the expression of a CAR molecule can also alter glucose metabolism in engineered T cells, and the specific CAR protein sequence alters the metabolic impact of CAR expression (139). Kawalekar et al (140) reported that anti-CD19 and mesothelin CARs containing the 4-1BB costimulatory domain promoted the differentiation of central memory T cells with enhanced respiratory capacity, mitochondrial biogenesis, and associated oxidative metabolism.…”